High-resolution detection of copy number alterations in single cells with HiScanner.

Improvements in single-cell whole-genome sequencing (scWGS) assays have enabled detailed characterization of somatic copy number alterations (CNAs) at the single-cell level. Yet, current computational methods are mostly designed for detecting chromosome-scale changes in cancer samples with low sequencing coverage. Here, we introduce HiScanner (High-resolution Single-Cell Allelic copy Number callER), which combines read depth, B-allele frequency, and haplotype phasing to identify CNAs with high resolution. In simulated data, HiScanner consistently outperforms state-of-the-art methods across various CNA types and sizes. When applied to high-coverage scWGS data from human brain cells, HiScanner shows a superior ability to detect smaller CNAs, uncovering distinct CNA patterns between neurons and oligodendrocytes. For 179 cells we sequenced from longitudinal meningioma samples, integration of CNAs with point mutations revealed evolutionary trajectories of tumor cells. These findings show that HiScanner enables accurate characterization of frequency, clonality, and distribution of CNAs at the single-cell level in both non-neoplastic and neoplastic cells.

Exome copy number variant detection, analysis, and classification in a large cohort of families with undiagnosed rare genetic disease.

Copy number variants (CNVs) are significant contributors to the pathogenicity of rare genetic diseases and, with new innovative methods, can now reliably be identified from exome sequencing. Challenges still remain in accurate classification of CNV pathogenicity. CNV calling using GATK-gCNV was performed on exomes from a cohort of 6,633 families (15,759 individuals) with heterogeneous phenotypes and variable prior genetic testing collected at the Broad Institute Center for Mendelian Genomics of the Genomics Research to Elucidate the Genetics of Rare Diseases consortium and analyzed using the seqr platform. The addition of CNV detection to exome analysis identified causal CNVs for 171 families (2.6%). The estimated sizes of CNVs ranged from 293 bp to 80 Mb. The causal CNVs consisted of 140 deletions, 15 duplications, 3 suspected complex structural variants (SVs), 3 insertions, and 10 complex SVs, the latter two groups being identified by orthogonal confirmation methods. To classify CNV variant pathogenicity, we used the 2020 American College of Medical Genetics and Genomics/ClinGen CNV interpretation standards and developed additional criteria to evaluate allelic and functional data as well as variants on the X chromosome to further advance the framework. We interpreted 151 CNVs as likely pathogenic/pathogenic and 20 CNVs as high-interest variants of uncertain significance. Calling CNVs from existing exome data increases the diagnostic yield for individuals undiagnosed after standard testing approaches, providing a higher-resolution alternative to arrays at a fraction of the cost of genome sequencing. Our improvements to the classification approach advances the systematic framework to assess the pathogenicity of CNVs.

Exome copy number variant detection, analysis and classification in a large cohort of families with undiagnosed rare genetic disease.

Copy number variants (CNVs) are significant contributors to the pathogenicity of rare genetic diseases and with new innovative methods can now reliably be identified from exome sequencing. Challenges still remain in accurate classification of CNV pathogenicity. CNV calling using GATK-gCNV was performed on exomes from a cohort of 6,633 families (15,759 individuals) with heterogeneous phenotypes and variable prior genetic testing collected at the Broad Institute Center for Mendelian Genomics of the GREGoR consortium. Each family's CNV data was analyzed using the seqr platform and candidate CNVs classified using the 2020 ACMG/ClinGen CNV interpretation standards. We developed additional evidence criteria to address situations not covered by the current standards. The addition of CNV calling to exome analysis identified causal CNVs for 173 families (2.6%). The estimated sizes of CNVs ranged from 293 bp to 80 Mb with estimates that 44% would not have been detected by standard chromosomal microarrays. The causal CNVs consisted of 141 deletions, 15 duplications, 4 suspected complex structural variants (SVs), 3 insertions and 10 complex SVs, the latter two groups being identified by orthogonal validation methods. We interpreted 153 CNVs as likely pathogenic/pathogenic and 20 CNVs as high interest variants of uncertain significance. Calling CNVs from existing exome data increases the diagnostic yield for individuals undiagnosed after standard testing approaches, providing a higher resolution alternative to arrays at a fraction of the cost of genome sequencing. Our improvements to the classification approach advances the systematic framework to assess the pathogenicity of CNVs.

Exome Sequencing and the Identification of New Genes and Shared Mechanisms in Polymicrogyria.

Importance: Polymicrogyria is the most commonly diagnosed cortical malformation and is associated with neurodevelopmental sequelae including epilepsy, motor abnormalities, and cognitive deficits. Polymicrogyria frequently co-occurs with other brain malformations or as part of syndromic diseases. Past studies of polymicrogyria have defined heterogeneous genetic and nongenetic causes but have explained only a small fraction of cases. Objective: To survey germline genetic causes of polymicrogyria in a large cohort and to consider novel polymicrogyria gene associations. Design, Setting, and Participants: This genetic association study analyzed panel sequencing and exome sequencing of accrued DNA samples from a retrospective cohort of families with members with polymicrogyria. Samples were accrued over more than 20 years (1994 to 2020), and sequencing occurred in 2 stages: panel sequencing (June 2015 to January 2016) and whole-exome sequencing (September 2019 to March 2020). Individuals seen at multiple clinical sites for neurological complaints found to have polymicrogyria on neuroimaging, then referred to the research team by evaluating clinicians, were included in the study. Targeted next-generation sequencing and/or exome sequencing were performed on probands (and available parents and siblings) from 284 families with individuals who had isolated polymicrogyria or polymicrogyria as part of a clinical syndrome and no genetic diagnosis at time of referral from clinic, with sequencing from 275 families passing quality control. Main Outcomes and Measures: The number of families in whom genetic sequencing yielded a molecular diagnosis that explained the polymicrogyria in the family. Secondarily, the relative frequency of different genetic causes of polymicrogyria and whether specific genetic causes were associated with co-occurring head size changes were also analyzed. Results: In 32.7% (90 of 275) of polymicrogyria-affected families, genetic variants were identified that provided satisfactory molecular explanations. Known genes most frequently implicated by polymicrogyria-associated variants in this cohort were PIK3R2, TUBB2B, COL4A1, and SCN3A. Six candidate novel polymicrogyria genes were identified or confirmed: de novo missense variants in PANX1, QRICH1, and SCN2A and compound heterozygous variants in TMEM161B, KIF26A, and MAN2C1, each with consistent genotype-phenotype relationships in multiple families. Conclusions and Relevance: This study's findings reveal a higher than previously recognized rate of identifiable genetic causes, specifically of channelopathies, in individuals with polymicrogyria and support the utility of exome sequencing for families affected with polymicrogyria.

Utility of Exome Sequencing for Diagnosis in Unexplained Pediatric-Onset Epilepsy.

Importance: Genomic advances inform our understanding of epilepsy and can be translated to patients as precision diagnoses that influence clinical treatment, prognosis, and counseling. Objective: To delineate the genetic landscape of pediatric epilepsy and clinical utility of genetic diagnoses for patients with epilepsy. Design, Setting, and Participants: This cohort study used phenotypic data from medical records and treating clinicians at a pediatric hospital to identify patients with unexplained pediatric-onset epilepsy. Exome sequencing was performed for 522 patients and available biological parents, and sequencing data were analyzed for single nucleotide variants (SNVs) and copy number variants (CNVs). Variant pathogenicity was assessed, patients were provided with their diagnostic results, and clinical utility was evaluated. Patients were enrolled from August 2018 to October 2021, and data were analyzed through December 2022. Exposures: Phenotypic features associated with diagnostic genetic results. Main Outcomes and Measures: Main outcomes included diagnostic yield and clinical utility. Diagnostic findings included variants curated as pathogenic, likely pathogenic (PLP), or diagnostic variants of uncertain significance (VUS) with clinical features consistent with the involved gene's associated phenotype. The proportion of the cohort with diagnostic findings, the genes involved, and their clinical utility, defined as impact on clinical treatment, prognosis, or surveillance, are reported. Results: A total of 522 children (269 [51.5%] male; mean [SD] age at seizure onset, 1.2 [1.4] years) were enrolled, including 142 children (27%) with developmental epileptic encephalopathy and 263 children (50.4%) with intellectual disability. Of these, 100 participants (19.2%) had identifiable genetic explanations for their seizures: 89 participants had SNVs (87 germline, 2 somatic mosaic) involving 69 genes, and 11 participants had CNVs. The likelihood of identifying a genetic diagnosis was highest in patients with intellectual disability (adjusted odds ratio [aOR], 2.44; 95% CI, 1.40-4.26), early onset seizures (aOR, 0.93; 95% CI, 0.88-0.98), and motor impairment (aOR, 2.19; 95% CI 1.34-3.58). Among 43 patients with apparently de novo variants, 2 were subsequently determined to have asymptomatic parents harboring mosaic variants. Of 71 patients who received diagnostic results and were followed clinically, 29 (41%) had documented clinical utility resulting from their genetic diagnoses. Conclusions and Relevance: These findings suggest that pediatric-onset epilepsy is genetically heterogeneous and that some patients with previously unexplained pediatric-onset epilepsy had genetic diagnoses with direct clinical implications.

A recurrent de novo variant in NUSAP1 escapes nonsense-mediated decay and leads to microcephaly, epilepsy, and developmental delay.

NUSAP1 encodes a cell cycle-dependent protein with key roles in mitotic progression, spindle formation, and microtubule stability. Both over- and under-expression of NUSAP1 lead to dysregulation of mitosis and impaired cell proliferation. Through exome sequencing and Matchmaker Exchange, we identified two unrelated individuals with the same recurrent, de novo heterozygous variant (NM_016359.5 c.1209C > A; p.(Tyr403Ter)) in NUSAP1. Both individuals had microcephaly, severe developmental delay, brain abnormalities, and seizures. The gene is predicted to be tolerant of heterozygous loss-of-function mutations, and we show that the mutant transcript escapes nonsense mediated decay, suggesting that the mechanism is likely dominant-negative or toxic gain of function. Single-cell RNA-sequencing of an affected individual's post-mortem brain tissue indicated that the NUSAP1 mutant brain contains all main cell lineages, and that the microcephaly could not be attributed to loss of a specific cell type. We hypothesize that pathogenic variants in NUSAP1 lead to microcephaly possibly by an underlying defect in neural progenitor cells.

Loss of non-motor kinesin KIF26A causes congenital brain malformations via dysregulated neuronal migration and axonal growth as well as apoptosis.

Kinesins are canonical molecular motors but can also function as modulators of intracellular signaling. KIF26A, an unconventional kinesin that lacks motor activity, inhibits growth-factor-receptor-bound protein 2 (GRB2)- and focal adhesion kinase (FAK)-dependent signal transduction, but its functions in the brain have not been characterized. We report a patient cohort with biallelic loss-of-function variants in KIF26A, exhibiting a spectrum of congenital brain malformations. In the developing brain, KIF26A is preferentially expressed during early- and mid-gestation in excitatory neurons. Combining mice and human iPSC-derived organoid models, we discovered that loss of KIF26A causes excitatory neuron-specific defects in radial migration, localization, dendritic and axonal growth, and apoptosis, offering a convincing explanation of the disease etiology in patients. Single-cell RNA sequencing in KIF26A knockout organoids revealed transcriptional changes in MAPK, MYC, and E2F pathways. Our findings illustrate the pathogenesis of KIF26A loss-of-function variants and identify the surprising versatility of this non-motor kinesin.

Biallelic loss of EMC10 leads to mild to severe intellectual disability.

The endoplasmic reticulum membrane protein complex subunit 10 (EMC10) is a highly conserved protein responsible for the post-translational insertion of tail-anchored membrane proteins into the endoplasmic reticulum in a defined topology. Two biallelic variants in EMC10 have previously been associated with a neurodevelopmental disorder. Utilizing exome sequencing and international data sharing we have identified 10 affected individuals from six independent families with five new biallelic loss-of-function and one previously reported recurrent EMC10 variants. This report expands the molecular and clinical spectrum of EMC10 deficiency, provides a comprehensive dysmorphological assessment and highlights an overlap between the clinical features of EMC10-and EMC1-related disease.

A recurrent, homozygous EMC10 frameshift variant is associated with a syndrome of developmental delay with variable seizures and dysmorphic features.

PURPOSE: The endoplasmic reticulum membrane complex (EMC) is a highly conserved, multifunctional 10-protein complex related to membrane protein biology. In seven families, we identified 13 individuals with highly overlapping phenotypes who harbor a single identical homozygous frameshift variant in EMC10. METHODS: Using exome, genome, and Sanger sequencing, a recurrent frameshift EMC10 variant was identified in affected individuals in an international cohort of consanguineous families. Multiple families were independently identified and connected via Matchmaker Exchange and internal databases. We assessed the effect of the frameshift variant on EMC10 RNA and protein expression and evaluated EMC10 expression in normal human brain tissue using immunohistochemistry. RESULTS: A homozygous variant EMC10 c.287delG (Refseq NM_206538.3, p.Gly96Alafs*9) segregated with affected individuals in each family, who exhibited a phenotypic spectrum of intellectual disability (ID) and global developmental delay (GDD), variable seizures and variable dysmorphic features (elongated face, curly hair, cubitus valgus, and arachnodactyly). The variant arose on two founder haplotypes and results in significantly reduced EMC10 RNA expression and an unstable truncated EMC10 protein. CONCLUSION: We propose that a homozygous loss-of-function variant in EMC10 causes a novel syndromic neurodevelopmental phenotype. Remarkably, the recurrent variant is likely the result of a hypermutable site and arose on distinct founder haplotypes.

Polymicrogyria is Associated With Pathogenic Variants in PTEN.

OBJECTIVE: Congenital structural brain malformations have been described in patients with pathogenic phosphatase and tensin homologue (PTEN) variants, but the frequency of cortical malformations in patients with PTEN variants and their impact on clinical phenotype are not well understood. Our goal was to systematically characterize brain malformations in patients with PTEN variants and assess the relevance of their brain malformations to clinical presentation. METHODS: We systematically searched a local radiology database for patients with PTEN variants who had available brain magnetic resonance imaging (MRI). The MRI scans were reviewed systematically for cortical abnormalities. We reviewed electroencephalogram (EEG) data and evaluated the electronic medical record for evidence of epilepsy and developmental delay. RESULTS: In total, we identified 22 patients with PTEN pathogenic variants for which brain MRIs were available (age range 0.4-17 years). Twelve among these 22 patients (54%) had polymicrogyria (PMG). Variants associated with PMG or atypical gyration encoded regions of the phosphatase or C2 domains of PTEN. Interestingly, epilepsy was present in only 2 of the 12 patients with PMG. We found a trend toward higher rates of global developmental delay (GDD), intellectual disability (ID), and motor delay in individuals with cortical abnormalities, although cohort size limited statistical significance. INTERPRETATION: Malformations of cortical development, PMG in particular, represent an under-recognized phenotype associated with PTEN pathogenic variants and may have an association with cognitive and motor delays. Epilepsy was infrequent compared to the previously reported high risk of epilepsy in patients with PMG. ANN NEUROL 2020;88:1153-1164.

Characterizing genomic alterations in cancer by complementary functional associations.

Systematic efforts to sequence the cancer genome have identified large numbers of mutations and copy number alterations in human cancers. However, elucidating the functional consequences of these variants, and their interactions to drive or maintain oncogenic states, remains a challenge in cancer research. We developed REVEALER, a computational method that identifies combinations of mutually exclusive genomic alterations correlated with functional phenotypes, such as the activation or gene dependency of oncogenic pathways or sensitivity to a drug treatment. We used REVEALER to uncover complementary genomic alterations associated with the transcriptional activation of ß-catenin and NRF2, MEK-inhibitor sensitivity, and KRAS dependency. REVEALER successfully identified both known and new associations, demonstrating the power of combining functional profiles with extensive characterization of genomic alterations in cancer genomes.

KRAS and YAP1 converge to regulate EMT and tumor survival.

Cancer cells that express oncogenic alleles of RAS typically require sustained expression of the mutant allele for survival, but the molecular basis of this oncogene dependency remains incompletely understood. To identify genes that can functionally substitute for oncogenic RAS, we systematically expressed 15,294 open reading frames in a human KRAS-dependent colon cancer cell line engineered to express an inducible KRAS-specific shRNA. We found 147 genes that promoted survival upon KRAS suppression. In particular, the transcriptional coactivator YAP1 rescued cell viability in KRAS-dependent cells upon suppression of KRAS and was required for KRAS-induced cell transformation. Acquired resistance to Kras suppression in a Kras-driven murine lung cancer model also involved increased YAP1 signaling. KRAS and YAP1 converge on the transcription factor FOS and activate a transcriptional program involved in regulating the epithelial-mesenchymal transition (EMT). Together, these findings implicate transcriptional regulation of EMT by YAP1 as a significant component of oncogenic RAS signaling.

ATARiS: computational quantification of gene suppression phenotypes from multisample RNAi screens.

Genome-scale RNAi libraries enable the systematic interrogation of gene function. However, the interpretation of RNAi screens is complicated by the observation that RNAi reagents designed to suppress the mRNA transcripts of the same gene often produce a spectrum of phenotypic outcomes due to differential on-target gene suppression or perturbation of off-target transcripts. Here we present a computational method, Analytic Technique for Assessment of RNAi by Similarity (ATARiS), that takes advantage of patterns in RNAi data across multiple samples in order to enrich for RNAi reagents whose phenotypic effects relate to suppression of their intended targets. By summarizing only such reagent effects for each gene, ATARiS produces quantitative, gene-level phenotype values, which provide an intuitive measure of the effect of gene suppression in each sample. This method is robust for data sets that contain as few as 10 samples and can be used to analyze screens of any number of targeted genes. We used this analytic approach to interrogate RNAi data derived from screening more than 100 human cancer cell lines and identified HNF1B as a transforming oncogene required for the survival of cancer cells that harbor HNF1B amplifications. ATARiS is publicly available at http://broadinstitute.org/ataris.

ß-Catenin-driven cancers require a YAP1 transcriptional complex for survival and tumorigenesis.

Wnt/ß-catenin signaling plays a key role in the pathogenesis of colon and other cancers; emerging evidence indicates that oncogenic ß-catenin regulates several biological processes essential for cancer initiation and progression. To decipher the role of ß-catenin in transformation, we classified ß-catenin activity in 85 cancer cell lines in which we performed genome-scale loss-of-function screens and found that ß-catenin active cancers are dependent on a signaling pathway involving the transcriptional regulator YAP1. Specifically, we found that YAP1 and the transcription factor TBX5 form a complex with ß-catenin. Phosphorylation of YAP1 by the tyrosine kinase YES1 leads to localization of this complex to the promoters of antiapoptotic genes, including BCL2L1 and BIRC5. A small-molecule inhibitor of YES1 impeded the proliferation of ß-catenin-dependent cancers in both cell lines and animal models. These observations define a ß-catenin-YAP1-TBX5 complex essential to the transformation and survival of ß-catenin-driven cancers.

Pivotal Advance: Th-1 cytokines inhibit, and Th-2 cytokines promote fibrocyte differentiation.

CD14+ peripheral blood monocytes can differentiate into fibroblast-like cells called fibrocytes, which are associated with and are at least partially responsible for wound healing and fibrosis in multiple organ systems. Signals regulating fibrocyte differentiation are poorly understood. In this study, we find that when added to human PBMCs cultured in serum-free medium, the profibrotic cytokines IL-4 and IL-13 promote fibrocyte differentiation without inducing fibrocyte or fibrocyte precursor proliferation. We also find that the potent, antifibrotic cytokines IFN-gamma and IL-12 inhibit fibrocyte differentiation. In our culture system, IL-1beta, IL-3, IL-6, IL-7, IL-16, GM-CSF, M-CSF, fetal liver tyrosine kinase 3, insulin growth factor 1, vascular endothelial growth factor, and TNF-alpha had no significant effect on fibrocyte differentiation. IL-4, IL-13, and IFN-gamma act directly on monocytes to regulate fibrocyte differentiation, and IL-12 acts indirectly, possibly through CD16-positive NK cells. We previously identified the plasma protein serum amyloid P (SAP) as a potent inhibitor of fibrocyte differentiation. When added together, the fibrocyte-inhibitory activity of SAP dominates the profibrocyte activities of IL-4 and IL-13. The profibrocyte activities of IL-4 and IL-13 and the fibrocyte-inhibitory activities of IFN-gamma and IL-12 counteract each other in a concentration-dependent manner. These results indicate that the complex mix of cytokines and plasma proteins present in inflammatory lesions, wounds, and fibrosis will influence fibrocyte differentiation.

Reaction kinetics of the addition of atomic sulfur to nitric oxide.

The reaction of S((3)P(J)) with NO ((2)Pi) in an Ar bath gas has been studied by the laser photolysis-resonance fluorescence technique over 300-810 K at pressures from 60 to 800 mbar. The observed second-order rate constants are close to the low-pressure limit. Fitting of Troe's formalism to experiment, with an estimated F(cent) = 0.78 exp(-T/7445) and k(infinity) given subsequently, yields k(0) = (6.2+/-0.6) x 10(-33) exp(+ (940+/-40)/T) cm(6) molecule(-2) s(-1). Error limits are +/-25%. A theoretical analysis of this value suggests that the average energy transferred during collisions between Ar and the excited intermediate is DeltaE = -360(-160) (+90) cm(-1). Over 300-800 K, the high-pressure limit is predicted to be k(infinity) = 2.2 x 10(-10) (T/300)(0.24) cm(3) molecule(-1) s(-1). Doublet and quartet adducts between S and NO were characterized via CBS-QB3 theory. The kinetic data can be rationalized with SNO ((2)A(')) as the major product, and an ab initio estimate of Delta(f)H(298) for SNO is 176+/-8 kJ mol(-1).